Antibiotic-induced modifications of the stiffness of bacterial membranes

In the latest years the importance of high resolution analysis of the microbial cell surface has been increasingly recognized. Indeed, in order to better understand bacterial physiology and achieve rapid diagnostic and treatment techniques, a thorough investigation of the surface modifications induced on bacteria by different environmental conditions or drugs is essential. Several instruments are nowadays available to observe at high resolution specific properties of microscopic samples. Among these, AFM can routinely study single cells in physiological conditions, measuring the mechanical properties of their membrane at a nanometric scale (force volume). Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work we exploit such technique to characterize bacterial systems. We have performed an analysis of the mechanical properties of bacteria (Escherichia coil) exposed to different conditions. Such measurements were performed on living bacteria, by changing in real-time the liquid environment: standard phosphate buffered saline, antibiotic (ampicillin) in PBS and growth medium. In particular we have focused on the determination of the membrane stiffness modifications induced by these solutions, in particular between stationary and replicating phases and what is the effect of the antibiotic on the bacterial structure. (C) 2013 Elsevier B.V. All rights reserved.

Force volume and stiffness tomography investigation on the dynamics of stiff material under bacterial membranes

Giovanni Dietler, Sandor Kasas, Giovanni Longo, Laura Rio Marques, Charles Roduit

The determination of the characteristics of micro-organisms in clinical specimens is essential for the rapid diagnosis and treatment of infections. A thorough investigation of the nanoscale properties of bacteria can prove to be a fundamental tool. Indeed, in the latest years, the importance of high resolution analysis of the properties of microbial cell surfaces has been increasingly recognized. Among the techniques available to observe at high resolution specific properties of microscopic samples, the Atomic Force Microscope (AFM) is the most widely used instrument capable to perform morphological and mechanical characterizations of living biological systems. Indeed, AFM can routinely study single cells in physiological conditions and can determine their mechanical properties with a nanometric resolution. Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work, we exploit the capabilities and peculiarities of AFM to analyze the mechanical properties of Escherichia coli in order to evidence with a high spatial resolution the mechanical properties of its structure. In particular, we will show that the bacterial membrane is not mechanically uniform, but contains stiffer areas. The force volume investigations presented in this work evidence for the first time the presence and dynamics of such structures. Such information is also coupled with a novel stiffness tomography technique, suggesting the presence of stiffer structures present underneath the membrane layer that could be associated with bacterial nucleoids. Copyright (C) 2012 John Wiley & Sons, Ltd.

Wiley-Blackwell2012

Molecular Modeling of Membrane Embedded Proteins

Thomas Lemmin

Over the past years, molecular modeling and simulation techniques have had a major impact on experimental life sciences. They are capable of providing accurate insight into microscopic mechanisms, which are usually difficult to investigate experimentally. Moreover, the integration of experimental data with molecular modeling appears to be a promising strategy to better understand complex biological processes. In this thesis, molecular dynamics simulation has been used in combination with experimental data to investigate two transmembrane proteins: (i) the bacterial chemoreceptor PhoQ and (ii) the Amyloid Precursor Protein (APP). (i) Bacterial two-component system PhoQ and bacterial membranes. Two-component systems (TCSs) are signaling complexes essential for bacterial survival and virulence. PhoQ is the histidine kinase chemoreceptor of the PhoQ-PhoP tandem machine that detects the concentration of cationic species at the inner membrane of Gram-negative bacteria. A full understanding of the PhoQ signal transduction mechanism is currently hindered by the lack of a complete atomistic structure. In this thesis project, the first structural model of the transmembrane (TM) portion of PhoQ from Escherichia coli was assembled, by using molecular simulations integrated with cross-linking disulfide scanning data. Its structural and dynamic features induce a concerted displacement of the TM helices at the periplasmic side, which modulates a rotation at the cytoplasmic end. This supports the idea that signal transduction is promoted through a combination of scissoring and rotational movements of the TM helices. Knowledge of this complex mechanism is essential in order to understand how the chemical stimuli sensed by the periplasmic sensor domain trigger, via the relay of the HAMP domain, the histidine auto-phosphorylation and kinase/phosphatase activity at the cytoplasmic end. The PhoQ sensor domain lies in close proximity to the membrane. Interaction with anionic lipids, such as phosphophatidylglycerols (PG) and cardiolipins (CL), are thought to play a key role in PhoQ activity. Present in bacterial and mitochondrial membranes, cardiolipins have a unique dimeric structure, which carries up to two charges, i.e. one per phosphate group, and under physiological conditions, can be unprotonated or singly protonated. Exhaustive models and characterization of cardiolipins are to-date scarce; therefore an ab initio parameterization of cardiolipin species for molecular simulation consistent with commonly used force fields is proposed here. Molecular dynamics (MD) simulations based on these models indicate a protonation-dependent lipid packing. A noteworthy interaction with solvating mono- and divalent cations is also observed. The proposed models will contribute to the biophysical and biochemical characterizations of bacterial and mitochondrial membranes and membrane-embedded proteins. (ii) Structural and dynamic properties of the Amyloid Precursor Protein. The Amyloid Precursor Protein (APP) is a type I membrane glycoprotein present at the neuronal synapsis. The proteolytic cleavage of its C-terminal segment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer"s disease (AD). Recently, the backbone structure of the APP transmembrane (TM) domain in detergent micelles was determined by nuclear magnetic resonance (NMR, independently by two different experimental groups). The TM conformations of these two structures are however markedly different. One is characterized by a highly kinked α-helix, whereas the other is mainly straight. Molecular dynamics simulations showed that the APP TM region is highly flexible and its secondary structure is influenced by the surrounding lipid environment. The size of the embedding detergent micelles strongly affects the conformation of the APP α-helix, with solvation being the main driving force for the development of a helical curvature. Once embedded in a membrane bilayer, APP systematically prefers a straight helical conformation. This is also confirmed when analyzing in silico the atomistic APP population observed in double electron-electron resonance (DEER) spectroscopy. In summary, the APP transmembrane domain is highly flexible due to the presence of glycine residues and can readily respond to the lipid environment, a property that might be critical for proteolytic processing by γ-secretase enzymes. The presented thesis work clearly shows how molecular simulations and their interplay with available experimental input can help advance the understanding of the mechanism of complex biological systems and processes on a molecular scale. These results, in particular, go well beyond the current understanding of the functioning of two transmembrane proteins relevant for human health. Furthermore, the computational approaches and procedures developed in these projects will hopefully promote novel integrated strategies for investigating biological systems.

EPFL2013

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

The physiological state of competence for natural transformation allows bacteria to take up free DNA from the environment and recombine it into their genome. As one of three modes of horizontal gene transfer, natural transformation has hence contributed substantially to the enormous diversity and plasticity of bacterial genomes. With its potential to promote the spread of antibiotic resistances and virulence factors of a variety of pathogens, natural transformation also has an important impact on human health. Even though conserved components required to undergo natural transformation have been identified in many bacterial species, our knowledge on the underlying mechanism of DNA transport remains very limited. To better understand this aspect, we established a first model of DNA uptake in the naturally competent human pathogen Vibrio cholerae. We showed that at least twenty proteins are required for efficient uptake, protection and recombination of transforming DNA in a non-species-specific manner. 14 of these proteins are needed to produce a type IV pilus, which we were able to visualize for the first time. We showed that this competence pilus is not restricted to cell poles, as has been shown for other competent bacterial species (e.g. Bacillus subtilis). Furthermore, we compared the position of this pilus to the localization of other competence proteins and determined their role in the assembly of a surface exposed pilus fiber. While this type IV pilus is important for efficient DNA uptake, we also observed rare transformation events in its absence. In contrast, three pilus-unrelated proteins that are likely involved in the transport process were absolutely required for transformation. Two of them were implicated in DNA transport across the inner membrane. Deletion of the corresponding genes led to a strong accumulation of transforming DNA within the periplasm. However, one of them, ComEA, was essential for DNA uptake across the outer membrane, independently of competence pili. We further characterized the role of this protein by showing that it is localized to the periplasm of competent V. cholerae cells and that it binds to DNA in vivo. Based on our results, we proposed that ComEA directly contributes to DNA transport. Although we showed that DNA uptake across the outer membrane and translocation of DNA into the cytoplasm are functionally uncoupled, our results suggest that the two events coincide spatially. In summary, these findings allow us to present a first mechanistic model of the DNA uptake process in V. cholerae. Many aspects of this model likely apply to other naturally competent bacteria, and therefore represent an important contribution to the general understanding of this basic biological process.